The present invention relates generally to the field of bioseparation and cell detection, and in particular to the field of biological sample processing to detect rare cells, and downstream applications for the purpose of screening for high risk population, diagnosing a disease, predicting disease or treatment outcome, monitoring a disease state or response to a therapy, optimizing a treatment regimen or developing a new therapy.
The mortality associated with malignant tumors is mostly due to the formation of metastasis in tissues and organs distant from the primary tumor. The early detection of the metastasis is a very important determinant of the probability of survival for cancer patients (Feldstein M, Zelen M, Inferring the natural time history of breast cancer: implications for tumor growth rate and early detection. Breast Cancer Res Treat. 1984; 4(1):3-10; Senie R T, Lesser M, Kinne D W, Rosen P P, Method of tumor detection influences disease-free survival of women with breast carcinoma, Cancer. 1994 Mar. 15; 73(6):1666-72; Carlson J A, Slominski A, Linette G P, Mysliborski J, Hill J, Mihm M C Jr, Ross J S, Malignant melanoma 2003: predisposition, diagnosis, prognosis, and staging. Am J Clin Pathol. 2003 December; 120 Suppl:S101-27).
Early detection of tumors and monitoring of tumor growth are considered a very critical element in the successful treatment of cancer patients. Current diagnostic technologies rely largely on imaging and histopathology. Various imaging and scanning methods allow the detection of tumor masses based on the differential metabolic activity or tissue density. Other imaging technologies such as colonoscopy and branchoscopy help identify tumor tissue by directly imaging through the surface of a lumen. These methodologies have a lower limit of resolution of a few millimeters, which is equivalent to a mass already containing a large number of tumor cells (>2×108). At this stage a tumor mass might be capable of shedding cancer cells in the bloodstream, with the potential to originate metastasis. Histopathology allows the diagnosis of a tumor utilizing a tissue sample obtained by means of a biopsy. While this approach provides direct visualization of the tumor cells, it's applicability in the diagnosis or monitoring of a tumor can be limited. Often, the location of the tumor is such that obtaining a biopsy sample may be impractical. In other cases, periodic monitoring by a temporal series of biopsy cannot be preformed at high enough frequency to provide useful information regarding the efficacy of a treatment and the progression of the disease. Another important limitation of the current histopathological approaches is that they can only provide data regarding the specific sites from which the biopsy was obtained, possibly overlooking the metastasis at other locations. Traditional histopathology can also be a risky process, during which tumor tissues or cells may be carried out to contaminate distal locations, causing potential metastasis during the biopsy procedure.
Typical progression of a localized tumor into a malignant cancer with metastasis involves multiple steps. At relatively early stages the tumor mass grows and the cells utilize nutrients available in the host tissue by simple diffusion. Later, once the tumor mass exceeds ˜1 mm in diameter, the tumor becomes vascularized, a step necessary for the tumor cells to have adequate supply of nutrients and retain their ability to grow. The vascularization step is driven in part by angiogenic factors released by the tumor cells. As the tumor growth progresses, the cells lose more and more of the properties of the tissue of origin including the ability to tightly interact with the neighboring cells (see for example Blanco M J, Moreno-Bueno G, Sarrio D, Locascio A, Cano A, Palacios J, Nieto M A, Correlation of Snail expression with histological grade and lymph node status in breast carcinomas. Oncogene. 2002 May 9; 21(20):3241-6). Eventually a small number of tumor cells will leak into the blood vessels that are in direct contact with the tumor. Once in circulation, the circulating tumor cells can be carried to distant sites in the body. The majority of these cells will not adapt to the environment of the bloodstream and will die while still in the circulation. However, occasionally a subset of cells may survive in the circulation for a longer time, by chance, or because of their more resilient property. When a circulating tumor cell enters the capillaries in a distal region, it may remain trapped at that location. A series of events may then take place for these cells to attach and translocate across the endothelial wall of the capillary. If the tumor cell survives the harsh environment in the blood stream until it crosses the capillary wall, it can invade the surrounding tissue and establish a metastasis at the new location.
As a general rule, the more aggressive a cancer grows and the more metastases it forms, the more difficult it will be to cure. Localized tumors can be treated by surgical removal or by chemotherapy or a combination of the two. However, once the cancer cells have established multiple colonies at different locations in the body, surgical interventions at many sites or on multiple organs in the body become impractical and of limited therapeutic value. A cancer characterized by a single or multiple metastasis may be treated with chemotherapy. However, even in this case the success of the therapy may be limited because the different cancer colonies (the primary tumor and the metastasis) often respond differently to any give chemotherapy treatment (El Hilali N, Rubio N, Blanco J. Different effect of paclitaxel on primary tumor mass, tumor cell contents, and metastases for four experimental human prostate tumors expressing luciferase. Clin Cancer Res. 2005 Feb. 1; 11(3):1253-8). The differential response to treatment of the different cancer colonies is attributed to two main factors: genetic heterogeneity of the cancer cells at the different sites and local environmental factors differentially affecting cancer cell survival at the various locations.
All of these considerations emphasize the importance of detecting metastasis at early stages. However, major challenges to early diagnosis are posed by the difficulty of detecting small metastases. A metastasis can be established starting from a single cell shed off by a primary tumor. Current imaging methodologies (X-ray, PET-scan, CT-scan) cannot provide early diagnosis of metastasis because their sensitivity in not sufficient to detect a single metastatic cell, but can only detect a metastasis once it grows to several millimeter in diameter and contains more than 100 million cells. The ability to detect cancer cells in circulation early on during the establishment of a metastasis would be of great clinical relevance. Although a tumor mass can shed a significant number of cells on a daily basis, the number of circulating cancer cells in any given sample of blood of clinically relevant volume (5 to 40 mL) is very low. These cells are often present in the blood at a frequency of one cancer cell per 107-108 white blood cells. This makes it very difficult to isolate and detect circulating cancer cells early in the metastatic process.
Tumors can occur in different tissues, such as epithelial and mesenchymal tissues. The majority of human solid tumors originate from epithelial cells that have undergone a transformation of their genetic material and their phenotype and escape the checkpoints that keep cell growth and cell division under control. It is generally recognized that a single mutation is not sufficient to generate a cancer cell but, instead, a sequence of mutations is necessary to initiate a tumor. The tumor cells are genetically unstable and continue to mutate and generate variants throughout the entire progression of the disease. The high genetic instability is the source of the heterogeneity observed among the tumor cells. In turn, the varied cell population in the tumor becomes the stage for evolutionary competition and selection in which the cells with the faster division time, highest metabolic rate and lowest differentiation grade, tend to grow and divide faster and eventually take over the entire population. Since new mutations and new tumor cell variants are constantly generated, a high degree of diversity is maintained in the tumor cell population.
The genetic instability and heterogeneity in the cells of the primary tumor has no clinical consequences since surgical intervention can remove the entire tumor mass and all of its cell variants. However, once the disease reaches the stage of metastatic cancer, the genetic diversity of the cancer cells in the primary tumor and the metastasis poses a major challenge for both the diagnosis as well as the treatment of the metastasis.
pithelial cells are tightly bound to one another and form sheets (called epithelia), which line all the cavities and surfaces of the body. One of the distinctive features of epithelial cells is the formation of tight intercellular junctions mediated by specialized protein complexes expressed on the surface of the epithelial cells. These cell-cell interactions leave very little space for extracellular space in epithelia and render the epithelia a selective barrier to the passage and diffusion of water, solutes and cells from one compartment of the body to another.
During the transformation of an epithelial cell into a metastatic cancer cell, numerous dramatic changes in the biological properties of the cell take place. The rate of growth and cell division is increased and the cell becomes progressively less dependent upon the growth control signal present in the normal tissue. Most importantly, in order to become metastatic, the cell needs to lose the key distinctive feature of epithelial cells: its ability to tightly bind to neighboring cells. In a well-known example of this situation, in breast cancer, the epithelial cell adhesion molecule E-Cadherin is lost in the cancer cells that progress to the metastatic stage. The protein E-Cadherin is actually known to be a “tumor suppressor”, that is, a factor whose higher expression tends to suppress the progression of cancer. The changes associated with the transformation of epithelial cells into tumor cells are so dramatic that the cancer cells cannot be identified as epithelial cells anymore. This transformation is often referred-to as epithelial-mesenchymal transition, to indicate the conversion of an epithelial cell, characterized by strong interactions with neighbors cells, into a mesenchymal cell, a cell with loose or no interaction with other cells and free to migrate in a tissue.
In recent years it has also became apparent that the metastases are likely originating from a small subpopulation of tumor cells called cancer stem cells. These cells may be present in the tumor mass at a frequency <2% and are less differentiated than the majority of the cells in the tumor. Cancer stem cells have gained the property of duplicating themselves in addition to producing a differentiated offspring cell, which allow them to become “immortal”, or having unlimited self reproduction potential. These cells have a high proliferation rate and can easily establish new metastasis at sites distant from the primary tumor. The importance of cancer stem cells in the diagnosis and treatment of metastatic cancer has been made clear by the observation that often the remission, or response to therapy as measured clinically using imaging of the primary tumor mass, is not predictive of the patient survival or time to recurrence. This apparent paradox can be explained by recognizing that the treatment capable (for example) of reducing the tumor mass, may be ineffective on the cancer stem cells, which are the main reason for uncontrolled and unlimited proliferation, and could still form distal metastasis.
At present, accumulating reports indicate that circulating tumor cells (CTCs) may be found in patients even before the primary tumor is detected. In addition to a potential role in early diagnosis and prognostication, CTCs may play a major role in characterizing genetic and immunophenotypic changes with tumor progression, thereby helping to guide individualized therapy. Though various techniques have been applied to isolate and characterize CTCs, many of them share the similar principle, i.e. antibody based positive selection. Apparently, application of this strategy for CTCs detection is limited by the availability, sensitivity, and specificity of antibodies against biomarkers on different tumor cells. Alternative strategies involve negative depletion of red blood cells (RBCs) and white blood cells (RBCs). The filtration approach to depletion the smaller RBCs and WBCs based on size risks losing target cells since certain target cells such as some CTCs can be as small as WBCs. Other approaches involving the lysis of RBCs might risk damaging target cells.
There is a need for a diagnostic methodology which has high sensitivity, and is capable of detecting cancer cells or cancer stem cells, or a certain population of more aggressive forms of cancer cells, such as cancer stem cells, in the body of a patient when said patient still has a relatively small tumor mass. In addition there is a need for a technology that can isolate and identify cancer cells or a certain population of more aggressive forms of cancer cells, such as cancer stem cells, in the body, regardless of the stage of the cancer, and the level of transformation accumulated by the cancer cells. Such a technology would allow for the identification of metastatic cancer disease at an early stage, or provide means for monitoring disease progression, response to therapy, or status of disease relapse. The present invention provides these and other benefits.